Hearing loss is a heterogeneous disorder that affects over 14 million people in the United States, with approximately 1 of every 1000 infants being affected by congenital deafness. An estimated one-half of congenital hearing loss cases are due to genetic causes (Bieber and Nance (1979) Clinical Geneticsxe2x80x94A Sourcebook for Physicians, Jackson and Schimke, eds., Wiley, N.Y., vol. 60, pp. 443-461). More than 175 different forms of hereditary deafness have been characterized, including autosomal dominant, autosomal recessive, X-linked, and mitochondrial forms (McKusick (1994) Mendelian Inheritance in Man, John Hopkins Univ. Press, Baltimore, Md.).
Genetic heterogeneity in hearing disorders both associated with other clinical anomalies (syndromic) and occurring as an isolated finding (nonsyndromic) indicates the involvement of a large number of genes in the complex development and function of the hearing process. Of the several hundred syndromic hearing loss disorders described (Gorlin et al. (1995) Hereditary Hearing Loss and Its Syndromes, Oxford Univ. Press, New York, N.Y.), only about 60 have been mapped to human chromosomes, with approximately half of these with characterized gene defects (Duyk et al., Nature Genet. 2:5-8, 1992; Petit, Nature Genet. 14:385-391, 1996). The majority of congenital hearing disorders are nonsyndromic (Cohen and Gorlin (1995) Hereditary Hearing Loss and its Syndromes, Gorlin, Toriello and Cohen, eds., Oxford Univ. Press, New York, N.Y., vol. 60, pp. 9-21), but even fewer nonsyndromic disorders have been identified. This number is increasing through the study of consanguineous geographically isolated families. Over 40 human chromosomal loci associated with nonsyndromic hearing impairment have been identified, some with corresponding mouse mutants in the homologous region (Petit (1996), supra; Van Camp et al., Am. J Hum. Genet. 60:758-764, 1997). However, to date, only a small number of nuclear genes responsible for nonsyndromic hearing impairment have been discovered. These include POU3F4 in DFN3 (de Kok et al. Science 267:685-688, 1995); MYO7A in DFNB2 (Liu et al., Nature Genet. 16:188-190, 1997; Weil et al. Nature Genet. 16:191-193, 1997) and DFN11 (Liu et al., Nature Genet. 17:268, 1997); POU4F3 in DFNA15 (Vahava et al., Science 279:1950, 1998); PDS in DFNB4 (Li et al.,Nature Genet18:215, 1998); TECTA in both DFNA8 and DFNA11 (Verhoeven et al., Nature Genet. 19:60, 1998); GJB2 in DFNB1 and DFNA3 (Kelsell et al., Nature 387:80-83, 1997).
The cause of many hearing disorders are still unknown. One such disorder is Meniere disease. Meniere disease is a syndrome in which hearing loss and imbalance problems co-occur. It is clinically characterized by recurrent episodes of vertigo associated with hearing loss and tinnitus with or without aural. Meniere disease is thought to be a multifactorial condition involving both genetic and environmental components.
The invention is based, at least in part, on the discovery that a nucleic acid and corresponding protein molecule (referred to herein as xe2x80x9cCOCHxe2x80x9d or xe2x80x9cCOCH5B2xe2x80x9d) are associated with the hearing disorder, Meniere disease. It was found that a missense mutation in the wild-type COCH5B2 gene, leads to an amino acid substitution in the COCH5B2 protein and plays a role in Meniere disease. Thus, mutated COCH5B2 molecules (also referred to as mtCOCH5B2 molecules) as described herein are useful in diagnosing hearing disorders such as Meniere disease. In addition, wild-type COCH5B2 (referred herein as xe2x80x9cCOCHxe2x80x9d or xe2x80x9cCOCH5B2xe2x80x9d) may be useful for the treatment of hearing disorders resulting from the expression and production of mtCOCH5B2. The properties of COCH5B2 are described in U.S. Ser. No. 09/394,264, which is incorporated herein by reference.
In general, the invention features a method of treating a subject at risk for (e.g., having or predisposed to) having Meniere disease. The method can include identifying an individual at risk for Meniere disease, and administering to the subject an effective amount of any of: COCH5B2 or a nucleic acid encoding it; an active fragment of COCH5B2 or a nucleic acid encoding it; an agonist of COCH5B2, e.g., a peptide or a peptomimetic analog; an antibody; or a small molecule, such that treatment of the subject occurs. Identification of an individual at risk for Meniere disease can be done in a number of different ways including: analyzing the family history of an individual; identifying an individual with vestibular dysfunction; identifying an individual by performing physical tests, e.g., otoscopy and pure tone audiometry; identifying an individual with a lesion in the COCH5B2 gene or mRNA; and/or identifying an individual with a mutant COCH5B2 protein sequence.
In a preferred embodiment, a method for treating a subject having Meniere disease comprises administering to the subject a COCH5B2 protein (SEQ ID NO:2), or portion thereof, such that treatment occurs. In another embodiment, Meniere disease can be treated by administering to the subject a nucleic acid encoding a COCH5B2 protein (SEQ ID NO:1 or SEQ ID NO3), or an active portion thereof, such that treatment occurs. The COCH5B2 molecule can be administered to a subject by any standard method, e.g., the COCH5B2 molecule can be administered by any of a number of different routes, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), or transmucosal administration.
In another aspect, the invention features a method of identifying an individual at risk for Meniere disease. The method includes detecting in the subject a genetic lesion(s) characterized by a mutation in the gene encoding a COCH5B2 protein (mtCOCH5B2 protein). In one embodiment, the lesion occurs in the COCH5B2 gene and a mutant mtCOCH5B2 protein is encoded. In another preferred embodiment, the lesion can be a deletion, insertion or substitution involving one or more nucleotides of the COCH5B2 gene, e.g., the lesion is a deletion or a substitution of nucleotide 151 of SEQ ID NO:3, e.g., the lesion is a substitution of the cytosine at nucleotide 151 of SEQ ID NO:3 to a thymidine. In another preferred embodiment, the lesion is a substitution, insertion or deletion of nucleotide 151 of SEQ ID NO:3, or a nucleotide which is 1, 2, 3, 5, 10 or more base-pairs on either side of nucleotide 151 of SEQ ID NO:3.
In one embodiment, the method includes contacting a sample, e.g., a cell sample, with a nucleic acid probe. In a preferred embodiment, the nucleic acid probe is capable of selectively binding a COCH5B2 nucleic acid sequence which contains a lesion, e.g., the probe only binds a COCH5B2 gene sequence which has a deletion, insertion or substitution. In a preferred embodiment, the probe binds a COCH5B2 sequence which has a substitution at nucleotide 151 of SEQ ID NO:3, e.g., the probe hybridizes to a COCH5B2 gene sequence that has a cytosine to thymidine substitution at nucleotide 151 of SEQ ID NO:3, e.g., the probe includes all or a portion of the nucleic acid sequence of SEQ ID NO:6 or the probe has the sequence 5xe2x80x2-tcctctgctcagggggc-3xe2x80x2 (SEQ ID NO:6).
In another embodiment, the method includes using a probe which can bind a mutant COCH5B2 protein which has a deletion, insertion or substitution at one or more amino acid residues. In a preferred embodiment, the probe can be a labeled probe or an antibody which is capable of selectively binding a mutant COCH5B2 protein. In another preferred embodiment, the labeled probe or antibody can selectively bind a COCH5B2 protein which has a substitution at amino acid residue 51 of SEQ ID NO:2, e.g., the proline at residue 51 of SEQ ID NO:2 is substituted with a serine; or the probe can selectively bind a mutant COCH5B2 protein which contains a deletion, insertion or substitution of 1, 2, 3, 4, 5 or more residues on either side of residue 51 of SEQ ID NO:2.
In a preferred embodiment, the method, as described above, can also be used in fetal or neonatal diagnosis.
Another aspect of the invention features a method for diagnosing a subject as having Meniere disease. The method includes contacting the subject or a sample (e.g., a cell or tissue sample, e.g., a biopsy sample) from the subject with an agent capable of selectively detecting a mutant form of the COCH5B2 protein. In a preferred embodiment, the agent is a labeled probe or an antibody that can bind a mutant COCH5B2 protein, e.g., the agent can bind a COCH5B2 protein which has a deletion, insertion or substitution in the COCH5B2 protein. In a preferred embodiment, the method uses a probe that selectively binds to a COCH5B2 protein which has a mutant residue at position 51 of SEQ ID NO:2, e.g., the proline at amino acid 51 of SEQ ID NO:2 is substituted for a serine.
In another embodiment, the method for diagnosing a subject as having MeniEre disease is based on detection of a genetic lesion in the COCH5B2 nucleic acid sequence. In a preferred embodiment, the method involves identifying an individual at risk for Meniere disease, contacting a sample (e.g., a cell or tissue sample, e.g., a biopsy sample) from the subject with an agent capable of detecting a mutation in the COCH5B2 gene sequence. In a preferred embodiment, the agent is a nucleic acid probe that can selectively bind a mutant COCH5B2 nucleic acid sequence, e.g., the probe can detect a mutation or deletion specific for Meniere disease, e.g., the probe can detect a mutation occurring at nucleotide 151 of SEQ ID NO:3, e.g., the mutation is a substitution of a cytosine at nucleotide 151 of SEQ ID NO:3 for a thymidine. In a preferred embodiment, the probe includes all or a portion of the nucleic acid sequence of SEQ ID NO:6 or the probe has the sequence 5xe2x80x2-tcctctgctcagggggc-3xe2x80x2 (SEQ ID NO:6). The method can also include comparing the sample from a subject at risk for Meniere disease to a control sample and forming a diagnosis based on whether a mutation is present as compared to the control sample. Specific diagnostic tests are described in greater detail below.
Another aspect of the invention features a kit for diagnosing a subject at risk for Meniere disease. The kit includes agents which can be used to detect whether a nucleic acid sequence from a subject of interest has a lesion in a COCH5B2 nucleic acid sequence or contains a mutant COCH5B2 protein. In one embodiment, a kit includes a nucleic acid probe which binds a COCH5B2 nucleic acid sequence which contains a lesion but not the wild type COCH5B2 sequence. In a preferred embodiment, the probe can bind a COCH5B2 nucleic acid sequence which contains a lesion, e.g., a COCH5B2 gene sequence which has a deletion, insertion or substitution. In a preferred embodiment, the probe selectively binds a COCH5B2 sequence which has a substitution at nucleotide 151 of SEQ ID NO:3, e.g., the probe selectively binds a COCH5B2 nucleic acid sequence where the cytosine at nucleotide 151 of SEQ ID NO:3 has been replaced with a thymidine e.g., a probe which includes all or a portion of the nucleic acid sequence of SEQ ID NO:6 or the probe which has the sequence 5xe2x80x2-tcctctgctcagggggc-3xe2x80x2 (SEQ ID NO:6). In another embodiment, the kit also includes a probe which selectively hybridizes to the wild-type COCH5B2 nucleic acid, e.g., the probe cannot bind a COCH5B2 containing a deletion, insertion or substitution. In a preferred embodiment, the probe cannot bind a COCH5B2 gene sequence which has a substitution at nucleotide 151 of SEQ ID NO:3 or a COCH5B2 sequence which has a deletion, insertion or substitution of 1, 2, 3, 5, 10 or more basepairs on either side of nucleotide 151 of SEQ ID NO:3. In still yet another embodiment, the kit also includes a probe that is capable of selectively binding a mutant COCH5B2 protein. The probe can be a labeled probe or an antibody that can selectively bind a COCH5B2 protein which has a substitution at amino acid residue 51 of SEQ ID NO:2, e.g., the proline at residue 51 of SEQ ID NO:2 is substituted with a serine; or the probe can selectively bind a mutant COCH5B2 protein which contains a deletion, insertion or substitution of 1, 2, 3, 4, 5 or more residues on either side of residue 51 of SEQ ID NO:2. In another embodiment, the kit also includes standards and controls, e.g., the kit includes both wild type and mutant COCH5B2 nucleic acids. In still yet another preferred embodiment, an instruction leaflet is enclosed which outlines how to use the components of the kit to diagnose an individual with Meniere disease.
Another aspect of the invention features an isolated nucleic acid molecule (e.g., cDNAs) comprising a nucleotide sequence encoding a mutant COCH5B2 protein or a biologically active portion thereof, as well as, nucleic acid fragments suitable as primers or hybridization probes for the detection of mutant COCH5B2-encoding nucleic acid (e.g., mRNA) and mutations thereof. In one embodiment, the isolated nucleic acid sequence includes the nucleotide sequence of SEQ ID NO:4, or the coding sequence or complement of these nucleotide sequences. In another preferred embodiment, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:5.
A preferred COCH5B2 nucleic acid sequence encodes a protein which possesses at least one of the mtCOCH5B2 activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein, or portion thereof, wherein the protein, or portion thereof, includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:5. Preferably, the protein, or a portion thereof, encoded by the nucleic acid molecule of the invention maintains the ability to play a disruptive role in inner ear biology, e.g., vestibular dysfunction, e.g., the protein or portion may cause hearing loss associated with one or more of the following symptoms: dizziness, balance problems, oscillopsia, vertigo, tinnitus, aural fullness, or nausea in an individual. For example, the protein can be involved in disrupting one or more of the following inner ear biology activities: 1) it can interact, e.g., bind, with components of the extracellular matrix (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); 2) it can modulate cell/extracellular matrix interactions; 3) it can modulate cell-cell adhesions; 4) it can interact, e.g., bind, with glycoproteins and/or proteoglycans for clearing them; 5) it can provide scaffolding by interacting with other extracellular matrix components (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); and 6) it can modulate an inner ear secretory pathway (e.g., it can modulate production of acidophilic deposits).
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4. More preferably, the isolated nucleic acid encodes a naturally-occurring mutant human COCH5B2. In a preferred embodiment, the isolated nucleic acid is at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 100, 150, 200 nucleotides in length. For example, the probe can include all or a portion of the nucleic acid sequence of SEQ ID NO:6 or the probe can have the sequence 5xe2x80x2-tcctctgctcagggggc-3xe2x80x2 (SEQ ID NO:6).
Other features and advantages of the invention will be apparent from the following detailed description and claims.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers to a mammal. Examples of mammals include human and non-human primates (e.g., a monkey), goats, pigs, cows, and rodents (e.g., a rat or a mouse) having a disorder associated with inner ear biology dysfunction, e.g., MeniEre disease. The mammal is preferably a primate, e.g., a human.
A xe2x80x9ctherapeutically effective amountxe2x80x9d refers to an amount which is capable of, at least partially, reducing, alleviating or preventing Meniere disease. A therapeutically effective amount can be determined on an individual basis and is based, at least in part, on consideration of the species of mammal, the mammal""s size, the agent used, the type of delivery system used, the time of administration relative to the severity of the disease, and whether a single, multiple, or a controlled release dose regimen is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.